Mullerian Inhibiting Substance (MIS) induces regression of the Mullerian duct in male embryos. The presence of MIS type II receptor in normal and neoplastic breast tissue suggests that breast is an additional target for MIS. MIS inhibited the growth of both estrogen receptor (ER) positive and negative breast cancer cells in vitro through interference with cell cycle progression and induction of apoptosis. This required activation of NFkB and was mediated in part by upregulation of its target gene IEX-IS (Segev et al. 2000). The importance of NFkappaB signaling in the breast is evident from its robust activation during post-lactational involution (Clarkson et al. 2000), the ability of NFkappaB p50/p65 dimers to inhibit beta-casein expression (Geymayer and Doppler, 2000), and its aberrant regulation in primary breast cancers and cancer cell lines (Sovak et al. 1997, Nakshatri et al. 1997). Thus using mice at various stages of breast development as an in vivo model system, we propose to test the hypothesis that MIS is a hormonal inhibitor of normal breast epithelial cell proliferation through activation of the NFkappaB pathway (Specific Aim 1). An inverse correlation between MIS type II receptor level and normal breast cell proliferation, and increased apoptosis of breast epithelial cells of female mice injected with MIS support the notion that MIS may be negative growth regulator of the breast. In this study, expression of MIS type II receptor and IEX-1S will be analyzed in the breast of a larger cohort of developing, virgin, pregnant, lactating and post-lactational rats. Further, mice in these stages of breast development will be injected with MIS and mammary tissue will be analyzed for activation of NFkappaB signaling, growth, and apoptosis. Primary human mammary epithelial cells will be used in vitro to directly link MIS- induced molecular signals as the causal events that lead to growth inhibition of normal mammary epithelial cells. To verify the hypothesis that aberration in MIS mediated growth inhibition in the breast leads to progression of cancer, human breast fibroadenomas and carcinomas will be screened for abnormalities in MIS type II receptor and IEX-1S (Specific Aim ll). Aberrant expression of these genes in tumors compared to normal tissue would predict that inability to respond to MIS-mediated signaling plays a role in neoplastic transformation of the breast. The utility of MIS in treatment of breast cancer will be evaluated by testing whether MIS inhibits the growth of human breast cancer cells grown in SCID mice (Specific Aim Ill). This study will identify whether MIS mediated signaling is a physiological suppressor of breast proliferation in the milieu of a number of growth factors which influence breast growth, and help us determine whether MIS will be of potential therapeutic benefit for both ER-positive and - negative breast cancer.